In general, the invention features novel methods for rapidly generating cell lines and mammals with site-specific genetic modifications of interest.
As the human genome sequencing effort approaches completion, much of the research focus has shifted from physical mapping of the genome to functional annotation of its contents. The challenge lies in evolving comprehensive approaches to efficiently define gene functions in vivo. The murine system is ideal for functional genomics because of the underlying biological similarity between human and mouse, the rapid advances in mouse genome sequencing, and the ability to genetically manipulate mouse embryonic stem (ES) cells.
Although gene disruption in mice has been widely used for functional analyses of genes in vivo, traditional procedures for generating site-specific gene knockout mice are generally time-consuming and labor-intensive. For these traditional methods, the targeting construct usually contains 6-10 kb of genomic sequence from the gene of interest with, e.g., a neomycin-resistant (neor) gene inserted into the coding region and a herpes-virus thymidine kinase (tk) gene placed at one end (FIG. 1A). The targeting construct is electroporated into ES cells and replaces the endogenous locus by homologous recombination. After verification by genomic DNA Blotting or by polymerase chain reaction (PCR) using primers that hybridize to the flanking region and the disrupted gene, ES cells bearing the mutant locus are injected into a mouse blastocyst to generate chimera mice. By either method, the flanking sequences of the replica cannot be too long. Finally, heterozygous and eventually homozygous mutant mice are obtained from the breeding of chimeric animals.
To make the targeting construct often requires fine restriction enzyme mapping of the gene and multi-step cloning, which is a long and tedious process. In order to prevent expression of a partial gene, which can result from alternative splicing, the insertion site should be as close to the translation initiation ATG as possible. This is often hindered by the lack of a convenient cloning site around the desired region. Furthermore, the length of homologous fragments that can be included in the targeting construct is often limited, resulting in low homologous recombination frequency. Data from haplotype mapping of human populations and studies of meiotic and mitotic recombination frequency in lower eukaryotes support the idea that favored sites for initiation of DNA exchange can be separated by up to several tens of kilobases from one another in euchromatin. If these findings are relevant to mitotic recombination in ES cells, it may be necessary to use very long flanking sequences to obtain high frequency homologous recombination. Creating such long flanking arms by conventional cloning is cumbersome and frequently impractical.
Thus, improved methods are needed to more efficiently generate genetically modified cell lines and non-human mammals.